Dendritic cells (DC) are known to play important roles in the induction of cellular immune responses against a variety of antigens (Ag), including chemical Ag, infectious pathogens, tumor-associated Ag, allo- Ag, and, more recently, auto-Ag. We hypothesize that experimental autoimmune reactions are inducible in skin, but only if one can load DC with relevant cutaneous Ag and only if one can break the tolerance against self-Ag. To overcome the first hurdle, we will generate DC hybrids fused with keratinocytes or fibroblasts. To overcome the second hurdle, we will engineer DC hybrids to secrete relatively large amounts of IL-12. Thus, our specific aims are: 1)To establish DC-keratinocyte and DC-fibroblast hybrids producing large amounts of IL-12, and 2) To characterize the cutaneous and systemic changes that are inducible in mice by immunization with DC hybrids. We have generated a HAT- sensitive, zeocin-resistant DC clone X5106-7 Zeo, which maintains phenotypic and functional properties of mature DC. We have also established the PEG-based fusion protocol and the HAT/zeocin-based selection protocol for DC hybrids. In the proposed experiments, XS 106- 7 Zeo DC clone will be fused with murine epidermal or dermal cells, and hybrid clones will first be characterized for their phenotype and function in vitro (Aim 1). The DC hybrid clones maintaining the original properties of both parental cell types will then be s.c. injected into otherwise naive mice to test their in vivo potentials to induce cellular and/or humoral immune reactions against self-Ag and to trigger clinical symptoms that are frequently associated with autoimmune skin diseases in human patients (Aim 2). We anticipate that the proposed studies will provide important insights into the pathogenesis of autoimmune diseases, the insights that may, ultimately, lead to the identification of auto-Ag and the development of new therapeutic strategies.